Compositions and methods for the treatment of carboxyhemoglobinemia

ABSTRACT

Described herein is a new antidote for the rapid elimination of carbon monoxide from hemoglobin, including brain, heart, and red cell hemoglobin. The disclosed therapy involves the use of modified human globins, particularly neuroglobins modified at residue 64 and cytoglobins modified at residue 81, which bind carbon monoxide with extremely high affinity. The monomeric mutant globins are infused into blood, where they rapidly and irreversibly sequester carbon monoxide, and thus limit toxic effects of carbon monoxide on cellular respiration and oxygen transport and utilization.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/834,035, filed Jun. 12, 2013, and U.S. Provisional Application No.61/799,155, filed Mar. 15, 2013. The above-listed applications areherein incorporated by reference in their entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numberHL103455 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD

This disclosure concerns recombinant globin molecules, such as mutantforms of neuroglobin and cytoglobin, that bind carbon monoxide with veryhigh affinity, and their use for the treatment of carboxyhemoglobinemiaand carbon monoxide poisoning.

BACKGROUND

Inhalation exposure to carbon monoxide represents a major cause ofenvironmental poisoning. Individuals can be exposed to carbon monoxidein the air under a variety of circumstances, such as house fires, use ofgenerators or outdoor barbeque grills used inside the house, or duringsuicide attempts by running automobiles in closed spaces. Carbonmonoxide binds to hemoglobin and to hemoproteins in cells, inparticular, the enzymes of the respiratory transport chain. Theaccumulation of carbon monoxide bound to hemoglobin and otherhemoproteins impairs oxygen delivery and oxygen utilization foroxidative phosphorylation. This ultimately results in severe hypoxic andischemic injury to vital organs such as the brain and the heart.Individuals who accumulate greater than 15% carbon carboxyhemoglobin intheir blood are at risk for brain injury and neurocognitive dysfunction.Individuals with higher levels of carboxyhemoglobin are at risk fordeath. Patients with very high carboxyhemoglobin levels typically sufferfrom irreversible brain injury and brain death.

Despite the availability of methods to rapidly diagnose carbon monoxidepoisoning with standard arterial and venous blood gas analysis andco-oximetry, and despite an awareness of risk factors for carbonmonoxide poisoning, there are no available antidotes for this toxicexposure. The current therapy is to give 100% oxygen by face mask, andwhen possible to expose patients to hyperbaric oxygen. The mechanism forhyperbaric oxygen therapy is the oxygen will increase the rate ofrelease of the carbon monoxide from hemoglobin and from tissues andaccelerate the natural clearance of carbon monoxide. However, thistherapy has only a modest effect on carbon monoxide clearance rates andbased on the complexity of hyperbaric oxygen facilities, this therapy isnot available in the field.

SUMMARY

A need exists for an effective, rapid and readily available therapy totreat carboxyhemoglobinemia, also known as carbon monoxide poisoning.Provided by the present disclosure are modified globin molecules, suchas recombinant forms of neuroglobin or cytoglobin, that bind carbonmonoxide with very high affinity, thereby functioning as carbon monoxidescavengers. The data disclosed herein demonstrates that the modifiedglobins can be used, for example, in methods of removing carbon monoxidefrom hemoglobin in blood or tissue, and in methods of treatingcarboxyhemoglobinemia.

Provided herein is a method of treating carboxyhemoglobinemia in asubject by selecting a subject with carboxyhemoglobinemia andadministering to the subject a therapeutically effective amount of arecombinant globin molecule that binds carbon monoxide with highaffinity. In some embodiments, the recombinant globin molecule is arecombinant human neuroglobin with a mutation at residue 64, such as aH64Q, H64L, H64A or H64W mutation, or a human recombinant cytoglobinwith a mutation at residue 81, such as a H81Q, H81A, H81L or H81Wmutation. In some examples, the recombinant globin molecules furthercomprise one or more cysteine amino acid substitutions to conferincreased solubility. Increased solubility allows for the production ofa high stock concentration of mutant globin for infusion of a dosesufficient for treatment of carboxyhemoglobinemia.

Also provided is a method of removing carbon monoxide from hemoglobin inblood or tissue by contacting the blood or tissue with a recombinantglobin molecule that binds carbon monoxide with high affinity. In someembodiments, the recombinant globin molecule is a recombinant humanneuroglobin with a mutation at residue 64, such as a H64Q, H64L, H64A orH64W mutation, or a human recombinant cytoglobin with a mutation atresidue 81, such as a H81Q, H81A, H81L or H81W mutation. In someexamples, the method of removing carbon monoxide from hemoglobin is anin vitro method. In other examples, the method is an in vivo method thatincludes administering the recombinant globin molecule to a subject inneed of treatment.

Further provided are human recombinant neuroglobin proteins comprising amutation at residue 64, and further comprising a C46G mutation, a C55Smutation and a C120S mutation. In some embodiments, the mutation atresidue 64 is a H64Q, H64A, H64L or H64W mutation. Similarly, thepresent disclosure provides human recombinant cytoglobin proteinscomprising a mutation at residue 81 and further comprising a C38Smutation and a C83S mutation. In some embodiments, the mutation atresidue 81 is a H81Q, H81A, H81L or H81W mutation. Compositionscomprising the recombinant neuroglobin or cytoglobin proteins and apharmaceutically acceptable carrier are also provided by the presentdisclosure.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are graphs showing the reaction of carboxylated red bloodcells (RBCs) with buffer or deoxy neuroglobin (deoxyNgb) H64Q. Theexperiments were performed at 21° C. in PBS buffer in anaerobicconditions. (FIG. 1A) Shown are deoxy or carboxylated species for theRBC fraction: deoxyHb during the RBC+Ngb experiment (diamonds); HbCOduring the RBC+Ngb experiment (squares); HbCO during the RBC+bufferexperiment (X); and deoxyHb during the RBC+buffer experiment(triangles). (FIG. 1B) Shown are deoxy or carboxylated species for thesupernatant (neuroglobin) fraction: deoxyNgb during the RBC+Ngbexperiment (diamonds); and NgbCO during the RBC+Ngb experiment(squares).

FIG. 2 is a graph showing the reaction of carboxylated red blood cellswith deoxyNgb H64Q. The experiment was performed at 21° C. in PBS bufferin anaerobic conditions. Shown are deoxy or carboxylated species for theRBC fraction: deoxyHb (diamonds); HbCO (squares); deoxyNgb (triangles);and NgbCO (X).

FIG. 3 is a graph showing the reaction of carboxylated red blood cellswith oxyNgb H64Q. The experiment was performed at 21° C. in PBS bufferin aerobic conditions. Shown are deoxy or carboxylated species for theRBC fraction: oxyHb (squares); HbCO (triangles); oxyNgb (X); NgbCO(asterisks).

FIG. 4 is a graph showing the concentration of HbCO in the blood ofCO-exposed mice. Mice were exposed to 1500 ppm CO for 60 minutes, thenCO was stopped and PBS (200 μl) was infused for 5 minutes. Blood samples(approximately 10 μl) were drawn every 5-10 minutes, chemically reducedwith dithionite and monitored for HbCO. Other Hb species in blood(oxyHb/metHb/deoxyHb) are represented as deoxy Hb.

FIG. 5 is a graph showing the concentration of HbCO in the blood ofCO-exposed mice. Mice were exposed to 1500 ppm CO for 70 minutes, afterwhich CO was stopped and concentrated H64Q Ngb (200 μl) was infused for10 minutes. Blood samples (about 10 μl) were drawn every 5-10 minutes,chemically reduced with dithionite and monitored for HbCO and NgbCO.Other Hb species in the blood (oxyHb/metHb/deoxyHb) are represented asdeoxy Hb, and Ngb species (oxyNgb/metNgb/deoxyNgb) are represented asdeoxy Ngb.

FIG. 6 is a graph showing the concentration of HbCO in the blood ofCO-exposed mice. Mice were exposed to 1500 ppm CO for 70 minutes, afterwhich CO was stopped and concentrated H64Q Ngb (200 μl) was infused for5 minutes. Blood samples (approximately 10 μl) were drawn every 5-10 or20 minutes, chemically reduced with dithionite and monitored for HbCOand NgbCO. Other Hb species in the blood (oxyHb/metHb/deoxyHb) arerepresented as deoxy Hb.

FIGS. 7A-7B are a pair of graphs showing decay of the concentration ofHbCO in the blood of CO-exposed mice after PBS or H64Q neuroglobininfusion. FIG. 7A shows absolute HbCO levels; FIG. 7B shows the relativechange in HbCO level. Mice were exposed to 1500 ppm CO for 60-70minutes, after which CO was stopped and concentrated H64Q Ngb (200 μl)was infused for 5 minutes. The infusion time was marked as t=0. Bloodsamples (about 10 μl) were drawn every 5-10 minutes, chemically reducedwith dithionite and monitored for HbCO and NgbCO. Other Hb species inthe blood (oxyHb/metHb/deoxyHb) are represented as deoxy Hb. Pointsrepresent the average and standard error of three or more experiments.

FIG. 8 is a graph showing percent decay of the concentration of HbCO inthe blood of CO-exposed mice 5 and 10 minutes after PBS or H64Qneuroglobin infusion. Bars represent from left to right: PBS infusionfor 5 minutes; PBS infusion for 10 minutes; H64Q Ngb infusion for 5minutes; and H64Q Ngb infusion for 10 minutes.

FIG. 9A is a graph demonstrating that H64Q neuroglobin is cleared asNgbCO in the urine of infused mice. Shown is the absorbance of urinefrom bladders of mice sacrificed approximately 75 minutes after the endof CO exposure. The top three traces show the absorbance due toneuroglobin, which include 82 to 91% NgbCO. The bottom traces indicatethe absorbance that is not attributable to Ngb. FIG. 9B is a photographof the internal organs of an H64Q Ngb-treated mouse and a syringe withred-colored urine containing NgbCO.

SEQUENCE LISTING

The amino acid sequences listed in the accompanying sequence listing areshown using standard three letter code for amino acids, as defined in 37C.F.R. 1.822. The Sequence Listing is submitted as an ASCII text file,created on Feb. 20, 2014, 9.98 KB, which is incorporated by referenceherein. In the accompanying sequence listing:

SEQ ID NO: 1 is an amino acid sequence of human neuroglobin.

SEQ ID NO: 2 is the amino acid sequence of a recombinant humanneuroglobin comprising one or more mutations.

SEQ ID NO: 3 is the amino acid sequence of a recombinant humanneuroglobin comprising mutations at residues 46, 55, 64 and 120.

SEQ ID NO: 4 is an amino acid sequence of human cytoglobin.

SEQ ID NO: 5 is the amino acid sequence of a recombinant humancytoglobin comprising one or more mutations.

SEQ ID NO: 6 is the amino acid sequence of a recombinant humancytoglobin comprising mutations at residues 38, 81 and 83.

DETAILED DESCRIPTION I. Abbreviations

Cgb cytoglobin

CO carbon monoxide

Hb hemoglobin

HbCO carboxyhemoglobin

Ngb neuroglobin

NgbCO carboxyneuroglobin

RBC red blood cell

II. Terms and Methods

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Administration: To provide or give a subject an agent, such as atherapeutic agent (e.g. a recombinant polypeptide), by any effectiveroute. Exemplary routes of administration include, but are not limitedto, injection or infusion (such as subcutaneous, intramuscular,intradermal, intraperitoneal, intrathecal, intravenous,intracerebroventricular, intrastriatal, intracranial and into the spinalcord), oral, intraductal, sublingual, rectal, transdermal, intranasal,vaginal and inhalation routes.

Antidote: An agent that neutralizes or counteracts the effects of apoison.

Carbon monoxide (CO): A colorless, odorless and tasteless gas that istoxic to humans and animals when encountered at sufficiently highconcentrations. CO is also produced during normal animal metabolism atlow levels.

Carboxyhemoglobin (HbCO): A stable complex of carbon monoxide (CO) andhemoglobin (Hb) that forms in red blood cells when CO is inhaled orproduced during normal metabolism.

Carboxyhemoglobinemia or carbon monoxide poisoning: A conditionresulting from the presence of excessive amounts of carbon monoxide inthe blood. Typically, exposure to CO of 100 parts per million (ppm) orgreater is sufficient to cause carboxyhemoglobinemia. Symptoms of mildacute CO poisoning include lightheadedness, confusion, headaches,vertigo, and flu-like effects; larger exposures can lead to significanttoxicity of the central nervous system and heart, and even death.Following acute poisoning, long-term sequelae often occur. Carbonmonoxide can also have severe effects on the fetus of a pregnant woman.Chronic exposure to low levels of carbon monoxide can lead todepression, confusion, and memory loss. Carbon monoxide mainly causesadverse effects in humans by combining with hemoglobin to formcarboxyhemoglobin (HbCO) in the blood. This prevents oxygen binding tohemoglobin, reducing the oxygen-carrying capacity of the blood, leadingto hypoxia. Additionally, myoglobin and mitochondrial cytochrome oxidaseare thought to be adversely affected. Carboxyhemoglobin can revert tohemoglobin, but the recovery takes time because the HbCO complex isfairly stable. Current methods of treatment for CO poisoning includingadministering 100% oxygen or providing hyperbaric oxygen therapy.

Contacting: Placement in direct physical association; includes both insolid and liquid form. When used in the context of an in vivo method,“contacting” also includes administering.

Cytoglobin: A globin molecule that is ubiquitously expressed in alltissues. Cytoglobin is a hexacoordinate hemoglobin that has beenreported to facilitate diffusion of oxygen through tissues, reducenitrite to nitric oxide, and play a cytoprotective role in hypoxicconditions and under oxidative stress. Human cytoglobin is 190 aminoacids in length. An exemplary human cytoglobin amino acid sequence isset forth herein as SEQ ID NO: 4. The recombinant cytoglobin mutantsdisclosed herein, which exhibit very high affinity for CO, comprise amutation at residue 81 (histidine to glutamine, alanine, tryptophan orleucine), and optionally comprise a cysteine to serine substitution atposition 38 and/or a cysteine to serine substitution at position 83 (seeSEQ ID NO: 5). In one non-limiting example, the cytoglobin mutant withhigh affinity for CO comprises the amino acid sequence of SEQ ID NO: 6.

Globin: A heme-containing protein involved in the binding and/ortransport of oxygen. Globins include, for example, hemoglobin,myoglobin, neuroglobin and cytoglobin.

Hemoglobin (Hb): The iron-containing oxygen-transport metalloprotein inthe red blood cells of the blood in vertebrates and other animals. Inhumans, the hemoglobin molecule is an assembly of four globular proteinsubunits. Each subunit is composed of a protein chain tightly associatedwith a non-protein heme group. Each protein chain arranges into a set ofalpha-helix structural segments connected together in a globin foldarrangement, so called because this arrangement is the same foldingmotif used in other heme/globin proteins. This folding pattern containsa pocket which strongly binds the heme group.

Neuroglobin (Ngb): A member of the globin family of proteins. Thephysiological function of neuroglobin is currently unknown, but isthought to provide protection under hypoxic or ischemic conditions.Neuroglobin is expressed in the central and peripheral nervous system,cerebral spinal fluid, retina and endocrine tissues. Human neuroglobinis 151 amino acids in length. An exemplary human neuroglobin sequence isprovided herein as SEQ ID NO: 1. The recombinant neuroglobin mutantsdisclosed herein, which exhibit very high affinity for CO, comprise amutation at residue 64 (histidine to glutamine, alanine, tryptophan orleucine), and optionally comprise a cysteine to glycine substitution atresidue 46, and/or a cysteine to serine substitution at position 55and/or a cysteine to serine substitution at position 120 (see SEQ ID NO:2). In one non-limiting example, the neuroglobin mutant with highaffinity for CO comprises the amino acid sequence of SEQ ID NO: 3.

Peptide or Polypeptide: A polymer in which the monomers are amino acidresidues which are joined together through amide bonds. When the aminoacids are alpha-amino acids, either the L-optical isomer or theD-optical isomer can be used, the L-isomers being preferred. The terms“peptide,” “polypeptide” or “protein” as used herein are intended toencompass any amino acid sequence and include modified sequences,including modified globin proteins. The terms “peptide” and“polypeptide” are specifically intended to cover naturally occurringproteins, as well as those which are recombinantly or syntheticallyproduced.

Conservative amino acid substitutions are those substitutions that, whenmade, least interfere with the properties of the original protein, thatis, the structure and especially the function of the protein isconserved and not significantly changed by such substitutions. Examplesof conservative substitutions are shown below.

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

Conservative substitutions generally maintain (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.

The substitutions which in general are expected to produce the greatestchanges in protein properties will be non-conservative, for instancechanges in which (a) a hydrophilic residue, for example, serine orthreonine, is substituted for (or by) a hydrophobic residue, forexample, leucine, isoleucine, phenylalanine, valine or alanine; (b) acysteine or proline is substituted for (or by) any other residue; (c) aresidue having an electropositive side chain, for example, lysine,arginine, or histidine, is substituted for (or by) an electronegativeresidue, for example, glutamine or aspartic acid; or (d) a residuehaving a bulky side chain, for example, phenylalanine, is substitutedfor (or by) one not having a side chain, for example, glycine.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition, 1975,describes compositions and formulations suitable for pharmaceuticaldelivery of the compositions disclosed herein.

In general, the nature of the carrier will depend on the particular modeof administration being employed. In addition to biologically neutralcarriers, pharmaceutical compositions to be administered can containminor amounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate.

Recombinant: A recombinant nucleic acid or protein is one that has asequence that is not naturally occurring or has a sequence that is madeby an artificial combination of two otherwise separated segments ofsequence. This artificial combination is often accomplished by chemicalsynthesis or by the artificial manipulation of isolated segments ofnucleic acids, for example, by genetic engineering techniques. The termrecombinant includes nucleic acids and proteins that have been alteredby addition, substitution, or deletion of a portion of a natural nucleicacid molecule or protein.

Sequence identity/similarity: The identity between two or more nucleicacid sequences, or two or more amino acid sequences, is expressed interms of the identity or similarity between the sequences. Sequenceidentity can be measured in terms of percentage identity; the higher thepercentage, the more identical the sequences are. Sequence similaritycan be measured in terms of percentage similarity (which takes intoaccount conservative amino acid substitutions); the higher thepercentage, the more similar the sequences are. Homologs or orthologs ofnucleic acid or amino acid sequences possess a relatively high degree ofsequence identity/similarity when aligned using standard methods. Thishomology is more significant when the orthologous proteins or cDNAs arederived from species which are more closely related (such as human andmouse sequences), compared to species more distantly related (such ashuman and C. elegans sequences).

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI) and onthe internet, for use in connection with the sequence analysis programsblastp, blastn, blastx, tblastn and tblastx. Additional information canbe found at the NCBI web site.

Subject: Living multi-cellular organisms, including vertebrateorganisms, a category that includes both human and non-human mammals.

Therapeutically effective amount: A quantity of compound or composition,for instance, a recombinant globin molecule, sufficient to achieve adesired effect in a subject being treated. For instance, this can be theamount necessary to scavenge carbon monoxide in the blood or tissues,reduce the level of HbCO in the blood, and/or reduce one or more signsor symptoms associated with carbon monoxide poisoning.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. “Comprising A or B” means including A, or B, or Aand B. It is further to be understood that all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent disclosure, suitable methods and materials are described below.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

III. Detailed Description

A need exists for an effective, rapid and readily available therapy totreat carboxyhemoglobinemia (including carbon monoxide poisoning).Disclosed herein is a new antidote that provides rapid elimination ofcarbon monoxide from the hemoglobin. The disclosed therapy involves theuse of modified human globins, particularly neuroglobins modified atresidue 64 (relative to SEQ ID NO: 1) and/or cytoglobins modified atresidue 81 (relative to SEQ ID NO: 4) that bind carbon monoxide withextremely high affinity. The data disclosed herein demonstrate for thefirst time that mutant globins with high affinity for CO can veryeffectively remove CO from red blood cells; the CO is then cleared fromthe system through secretion in the urine. Thus, the mutant globinmolecules described herein provide a surprisingly effective treatmentfor carboxyhemoglobinemia. The monomeric mutant globins are, forexample, infused into blood in a subject, where they rapidly andirreversibly sequester carbon monoxide, and thus limit toxic effects ofcarbon monoxide on cellular respiration and oxygen transport andutilization.

A. Methods of Treating Carboxyhemoglobinemia or CO Poisoning

Provided herein is a method of treating carboxyhemoglobinemia in asubject by selecting a subject with carboxyhemoglobinemia andadministering to the subject a therapeutically effective amount of arecombinant globin molecule that binds carbon monoxide with highaffinity. In some embodiments, the recombinant globin molecule is arecombinant human neuroglobin with a mutation (an amino acidsubstitution) at residue 64, such as a H64Q, H64L, H64A or H64Wmutation, or a human recombinant cytoglobin with a mutation at residue81, such as a H81 Q, H81A, H81L or H81W mutation. Throughout thisdisclosure, the amino acid positions of human recombinant neuroglobinand human recombinant cytoglobin are based on the wild-type humanneuroglobin and wild-type human cytoglobin sequences set forth herein asSEQ ID NO: 1 and SEQ ID NO: 4, respectively.

The recombinant globin molecules can further include one or morecysteine amino acid substitutions to confer increased solubility.Increased solubility allows for the production of a high stockconcentration of mutant globin for infusion of a dose sufficient fortreatment of carboxyhemoglobinemia. In some embodiments, humanrecombinant neuroglobin further comprises a C46G mutation, a C55Smutation, a C120S mutation, or any combination thereof. In particularexamples, the recombinant neuroglobin comprises all three cysteinesubstitutions. In one non-limiting example, the human recombinantneuroglobin comprises the amino acid sequence of SEQ ID NO: 3. In someembodiments, human recombinant cytoglobin further comprises a C38Smutation, or a C83S mutation, or both a C38S and a C83S mutation. In onenon-limiting example, the human recombinant cytoglobin comprises theamino acid sequence of SEQ ID NO: 6.

In alternative embodiments, the human recombinant neuroglobin comprisinga mutation at residue 64, and optionally one, two or three cysteinesubstitutions (i.e., one, two or all three of the C46G, C55S and C120Smutations) can include one or more conservative or non-conservativeamino acid substitutions at other residues. Similarly, the humanrecombinant cytoglobin comprising a mutation at residue 81, andoptionally one or both of the cysteine substitutions (i.e. one or bothof the C38S and C83S mutations) can include one or more conservative ornon-conservative amino acid substitutions at other residues. In someexamples, the human recombinant neuroglobin or cytoglobin comprises one,two, three, four, five, six, seven, eight, nine or ten conservativeamino acid substitutions, or one, two, three, four, five, six, seven,eight, nine or ten non-conservative amino acid substitutions, or anycombination of conservative and non-conservative substitutions, as longas the recombinant globin retains the capacity to bind carbon monoxidewith high affinity. In some examples, the recombinant human neuroglobinor cytoglobin includes a deletion, such as a deletion of one, two,three, four, five, six, seven, eight, nine, ten or more amino acids,while still maintaining the capacity to bind carbon monoxide with highaffinity.

In some examples, the human recombinant neuroglobin comprising amutation at residue 64, and optionally one, two or three cysteinesubstitutions (i.e., one, two or all three of the C46G, C55S and C120Smutations), is at least 85%, at least 90%, at least 95%, at least 96%,at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1 orSEQ ID NO: 3.

In some examples, the human recombinant cytoglobin comprising a mutationat residue 81, and optionally one or both of the cysteine substitutions(i.e. one or both of the C38S and C83S mutations) is at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% identical to SEQ ID NO: 4 or SEQ ID NO: 6.

In some embodiments, the subject has at least 3%, at least 5%, at least10%, at least 15% or at least 20% carboxyhemoglobin (HbCO) in theirblood.

In some embodiments, the recombinant globin molecule is administeredintravenously, such as by intravenous infusion.

An appropriate dose of recombinant neuroglobin or cytoglobin can bedetermined by a medical practitioner. In some embodiments, the dose isthe amount of recombinant globin required to decrease HbCO at least 1%,at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80% or at least 90%.Generally, a dose of about 136 grams of neuroglobin or about 184 gramsof cytoglobin is sufficient to achieve a 20% reduction in HbCO.

Thus, in some embodiments, the therapeutically effective dose of humanrecombinant neuroglobin is about 25 to about 1000 grams, about 50 toabout 500 grams, about 50 to about 200 grams, or about 60 to about 140grams. In particular examples, the therapeutically effective dose ofhuman recombinant neuroglobin is about 50, about 75, about 100, about150, about 200, about 250, about 300, about 350, about 400, about 450 orabout 500 grams. In some embodiments, the therapeutically effective doseof human recombinant cytoglobin is about 25 to about 1000 grams, orabout 50 to about 800 grams. In particular examples, the therapeuticallyeffective does of human recombinant cytoglobin is about 50, about 75,about 100, about 150, about 200, about 250, about 300, about 350, about400, about 450, about 500, about 550, about 600, about 650, about 700,about 750 or about 800 grams.

In some embodiments, the neuroglobin or cytoglobin concentrationadministered to a subject is about 70 to about 200 grams per liter,which equates to approximately 35-200 grams for a 500 milliliter or 1liter treatment.

The modified globin with high affinity for carbon monoxide can beadministered to a subject in a single dose, or in multiple doses asneeded, to reduce HbCO to a non-toxic level.

In some embodiments, the dose administered to the subject is the amountof recombinant globin required to decrease HbCO by at least 1%, at least2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80% or at least 90% (compared to thelevel of HbCO before treatment) in blood and/or tissue of the subject.

B. Methods of Removing Carbon Monoxide from Hemoglobin

Also provided herein is a method of removing carbon monoxide fromhemoglobin in blood or tissue by contacting the blood or tissue with arecombinant globin molecule that binds carbon monoxide with highaffinity. In the context of the present disclosure, “removing” does notrequire complete elimination of carbon monoxide from the blood ortissue, but rather means removal of CO from hemoglobin molecules inblood or tissue such that the overall level of HbCO is reduced in theblood or tissue of a sample or subject. For example, the HbCO can bereduced by at least 1%, at least 2%, at least 3%, at least 4%, at least5%, at least 10%, at least 15%, at least 20% or at least 25% (comparedto the level of HbCO before treatment) in the blood and/or tissue.

In some embodiments of the method, the recombinant globin molecule is arecombinant human neuroglobin with a mutation at residue 64, such as aH64Q, H64L, H64A or H64W mutation, or a human recombinant cytoglobinwith a mutation at residue 81, such as a H81 Q, H81A, H81L or H81Wmutation.

The recombinant globin molecules can further include one or morecysteine amino acid substitutions to confer increased solubility.Increased solubility allows for the production of a high stockconcentration of mutant globin for infusion of a dose sufficient foreffective removal of CO from the blood. In some embodiments, humanrecombinant neuroglobin further comprises a C46G mutation, a C55Smutation, a C120S mutation, or any combination thereof. In particularexamples, the recombinant neuroglobin comprises all three cysteinesubstitutions. In one non-limiting example, the human recombinantneuroglobin comprises the amino acid sequence of SEQ ID NO: 3. In someembodiments, human recombinant cytoglobin further comprises a C38Smutation, or a C83S mutation, or both a C38S and a C83S mutation. In onenon-limiting example, the human recombinant cytoglobin comprises theamino acid sequence of SEQ ID NO: 6.

In alternative embodiments, the human recombinant neuroglobin comprisinga mutation at residue 64, and optionally one, two or three cysteinesubstitutions (i.e., one, two or all three of the C46G, C55S and C120Smutations) can include one or more conservative or non-conservativeamino acid substitutions at other residues. Similarly, the humanrecombinant cytoglobin comprising a mutation at residue 81, andoptionally one or both of the cysteine substitutions (i.e. one or bothof the C38S and C83S mutations) can include one or more conservative ornon-conservative amino acid substitutions at other residues. In someexamples, the human recombinant neuroglobin or cytoglobin comprises one,two, three, four, five, six, seven, eight, nine or ten conservativeamino acid substitutions, or one, two, three, four, five, six, seven,eight, nine or ten non-conservative amino acid substitutions, or anycombination of conservative and non-conservative substitutions, as longas the recombinant globin retains the capacity to bind carbon monoxidewith high affinity. In some examples, the recombinant human neuroglobinor cytoglobin includes a deletion, such as a deletion of one, two,three, four, five, six, seven, eight, nine, ten or more amino acids,while still maintaining the capacity to bind carbon monoxide with highaffinity.

In some examples, the human recombinant neuroglobin comprising amutation at residue 64, and optionally one, two or three cysteinesubstitutions (i.e., one, two or all three of the C46G, C55S and C120Smutations), is at least 85%, at least 90%, at least 95%, at least 96%,at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1 orSEQ ID NO: 3.

In some examples, the human recombinant cytoglobin comprising a mutationat residue 81, and optionally one or both of the cysteine substitutions(i.e. one or both of the C38S and C83S mutations) is at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% identical to SEQ ID NO: 4 or SEQ ID NO: 6.

In some examples, the method of removing carbon monoxide from hemoglobinis an in vitro method. For example, the method can include contacting ablood or tissue sample with the recombinant globin molecule. In someexamples, a sufficient amount of recombinant globin is contacted withthe blood or tissue sample such that HbCO is reduced in the sample by atleast 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80% or at least 90%.

In other examples, the method is an in vivo method that includesadministering the recombinant globin molecule to a subject in need oftreatment. In some cases, the subject (prior to administration) has atleast 3%, at least 5%, at least 10%, at least 15% or at least 20% HbCOin their blood. The recombinant globin molecule can be administeredusing any suitable route of administration, such as intravenousadministration, for example intravenous infusion.

Appropriate, therapeutically effective doses of recombinant neuroglobinand cytoglobin are discussed above in section A. In some examples, thetherapeutically effective dose of human recombinant neuroglobin is about25 to about 1000 grams, or about 50 to about 500 grams. In someexamples, the therapeutically effective dose of human recombinantcytoglobin is about 25 to about 1000 grams, or about 50 to about 800grams. The modified globin with high affinity for carbon monoxide can beadministered to a subject in a single dose, or in multiple doses asneeded, to reduce HbCO to a non-toxic level.

In some embodiments of the in vivo method, the dose administered to thesubject is the amount of recombinant globin required to decrease HbCO atleast 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80% or at least 90% in bloodand/or tissue of the subject.

C. Recombinant Neuroglobin and Cytoglobin Mutants

Provided herein are human recombinant neuroglobin proteins comprising amutation at residue 64 (relative to SEQ ID NO: 1), and furthercomprising a C46G mutation, a C55S mutation and a C120S mutation. Insome embodiments, the mutation at residue 64 is a H64Q, H64A, H64L orH64W mutation. Similarly, the present disclosure provides humanrecombinant cytoglobin proteins comprising a mutation at residue 81(relative to SEQ ID NO: 4) and further comprising a C38S mutation and aC83S mutation. In some embodiments, the mutation at residue 81 is aH81Q, H81A, H81L or H81W mutation. Further provided are compositionscomprising the recombinant neuroglobin or cytoglobin proteins disclosedherein and a pharmaceutically acceptable carrier.

Data disclosed herein (see Example 2) demonstrates for the first timethat neuroglobin molecules comprising the H64L or H64Q mutation haveauto-oxidation rates 23-fold slower than the wild type protein. A lowerauto-oxidation rate is a desirable property because it reduces sidereactions that are detrimental to the ability of the mutant globin tobind CO and deliver oxygen to tissues.

Wild-type human neuroglobin and wild-type human cytoglobin amino acidsequences are shown below, along with exemplary recombinant neuroglobinand cytoglobin mutants provided by the present disclosure.

Human neuroglobin (SEQ ID NO: 1; GenBank™ Accession No. NP_067080.1)MERPEPELIRQSWRAVSRSPLEHGTVLFARLFALEPDLLPLFQYNCRQFSSPEDCLSSPEFLDHIRKVMLVIDAAVTNVEDLSSLEEYLASLGRKHRAVGVKLSSFSTVGESLLYMLEKCLGPAFTPATRAAWSQLYGAVV QAMSRGWDGERecombinant neuroglobin with high affinity for CO (SEQ ID NO: 2)MERPEPELIRQSWRAVSRSPLEHGTVLFARLFALEPDLLPLFQYNX₁RQFSSPEDX₂LSSPEFLDX₃IRKVMLVIDAAVTNVEDLSSLEEYLASLGRKHRAVGVKLSSFSTVGESLLYMLEKX₂LGPAFTPATRAAWSQLY GAVVQAMSRGWDGE X₁ =C or G X₂ = C or S X₃ = Q, L, A or WRecombinant neuroglobin with four amino acid substitutions(SEQ ID NO: 3) MERPEPELIRQSWRAVSRSPLEHGTVLFARLFALEPDLLPLFQYNGRQFSSPEDSLSSPEFLDQIRKVMLVIDAAVTNVEDLSSLEEYLASLGRKHRAVGVKLSSFSTVGESLLYMLEKSLGPAFTPATRAAWSQLYGAVVQAM SRGWDGEHuman cytoglobin (SEQ ID NO: 4; GenBank™ Accession No. NP_599030)MEKVPGEMEIERRERSEELSEAERKAVQAMWARLYANCEDVGVAILVRFFVNFPSAKQYFSQFKHMEDPLEMERSPQLRKHACRVMGALNTVVENLHDPDKVSSVLALVGKAHALKHKVEPVYFKILSGVILEVVAEEFASDFPPETQRAWAKLRGLIYSHVTAAYKEVGWVQQVPNATTPPATLPSSGPRecombinant cytoglobin with high CO affinity (SEQ ID NO: 5)MEKVPGEMEIERRERSEELSEAERKAVQAMWARLYANX₁EDVGVAILVRFFVNFPSAKQYFSQFKHMEDPLEMERSPQLRIOC₂AX₁RVMGALNTVVENLHDPDKVSSVLALVGKAHALKHKVEPVYFKILSGVILEVVAEEFASDFPPETQRAWAKLRGLIYSHVTAAYKEVGWVQQVPNATTPPAT LPSSGP X₁ = C or S X₂ =Q, L, A or W Recombinant cytoglobin with three amino acid substitutions(SEQ ID NO: 6) MEKVPGEMEIERRERSEELSEAERKAVQAMWARLYANSEDVGVAILVRFFVNFPSAKQYFSQFKHMEDPLEMERSPQLRKQASRVMGALNTVVENLHDPDKVSSVLALVGKAHALKHKVEPVYFKILSGVILEVVAEEFASDFPPETQRAWAKLRGLIYSHVTAAYKEVGWVQQVPNATTPPATLPSS GP

The human recombinant neuroglobin and cytoglobin proteins providedherein can comprises one of the sequences shown above (and set forthherein as SEQ ID NOs: 2, 3, 5 and 6), or the recombinant globins canfurther include one or more conservative or non-conservative amino acidsubstitutions. For example, the human recombinant neuroglobin withmutations at residues 46, 55, 64 and 120 can include one or moreconservative or non-conservative amino acid substitutions at otherresidues. Similarly, the human recombinant cytoglobin comprisingmutations at residues 38, 81 and 83 can include one or more conservativeor non-conservative amino acid substitutions at other residues. In someexamples, the human recombinant neuroglobin or cytoglobin comprises one,two, three, four, five, six, seven, eight, nine or ten conservativeamino acid substitutions, or one, two, three, four, five, six, seven,eight, nine or ten non-conservative amino acid substitutions, or anycombination of conservative and non-conservative substitutions, as longas the recombinant globin retains the capacity to bind carbon monoxidewith high affinity. In some examples, the recombinant human neuroglobinor cytoglobin includes a deletion, such as a deletion of one, two,three, four, five, six, seven, eight, nine, ten or more amino acids,while still maintaining the capacity to bind carbon monoxide with highaffinity.

In some examples, the human recombinant neuroglobin is at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% identical to SEQ ID NO: 2 or SEQ ID NO: 3 and retains themodification at residue 64 (H64Q, H64A, H64W or H64L) and the C46G, C55Sand C120S mutations.

In some examples, the human recombinant cytoglobin is at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% identical to SEQ ID NO: 6 and retains the modification atresidue 81 (H81Q, H81A, H81W or H81L) and the C38S and C83S mutations.

Compositions comprising any of the recombinant globin moleculesdisclosed herein and a pharmaceutically acceptable carrier, are alsoprovided by the present disclosure.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1 Scavenging of Carbon Monoxide (CO) from Hemoglobin bythe Neuroglobin H64Q Mutant

This example demonstrates that H64Q mutant neuroglobin rapidly removesCO from carboxylated hemoglobin located inside red blood cells.

Background

Neuroglobin (Ngb) is a heme protein, recently discovered in mammals andother species (Burmester et al., Nature 407(6803):520-523, 2000). Ngb isvery similar in sequence and structure to myoglobin and hemoglobin, butunlike these proteins it contains a six-coordinated heme with twohistidine groups binding to the heme, whereas myoglobin and hemoglobinare five-coordinated and only have one histidine permanently bound tothe heme. The function of this heme protein is unknown. The reaction ofthe iron atom from a heme group can be depicted as follows:

where k_(on) and k_(off) are the rates of CO binding and dissociation,respectively. Ngb shows high affinity for ligands such as oxygen orcarbon monoxide, and this affinity is even higher when the distalhistidine (His64) is replaced by other side chains (Table 1).

TABLE 1 Binding and dissociation constants for neuroglobin andhemoglobin k_(on) k_(off) (M⁻¹s⁻¹) (s⁻¹) Human Ngb wt  65 × 10⁶ 0.014Mouse Ngb wt  72 × 10⁶ 0.013 Mouse Ngb H64L  200 × 10⁶ ND (too slow)Human Hb (α subunit, R-state)   6 × 10⁶ 0.012 Human Hb (α subunit,T-state) 0.12 × 10⁶ 0.21 Human Hb (β subunit, R-state)  7.4 × 10⁶ 0.007Human Hb (β subunit, T-state) 0.05 × 10⁶ 0.19 Values determined at 25°C. Neuroglobin data from Dewilde et al., J Biol Chem 276(42):38949-38955, 1998. Hemoglobin data from Unzai et al., J Biol Chem273(36): 23150-23159, 1998.

Based on previous characterization (Tiso et al., J Biol Chem286(20):18277-18289, 2011) and subsequent studies, it is believed thatthe CO binding properties of the H64Q and H64L mutants are very similar,therefore the reported values for H64L are a reasonable estimate forH64Q. The data in Table 1 indicates that in the presence of an adequatescavenger, the CO dissociation from hemoglobin will lead to a half-lifeof the HbCO complex of approximately 3.5 seconds (T-state) or 70 seconds(R-state) at 25° C. Higher rates may lead to even shorter times at 37°C. These short times offer an opportunity for a therapeutic approach.However, in the absence of CO scavengers, the dissociated CO willeventually bind again to hemoglobin, leading to a persistence of HbCOmuch longer than suggested by the dissociation rates.

The binding and dissociation rates for neuroglobin (Table 1) indicate amuch higher affinity towards CO than that of hemoglobin. Therefore, itwas hypothesized that neuroglobin would be a suitable scavenger of COfrom HbCO or other carboxylated compounds. The results described belowconfirm this hypothesis.

Materials and Methods Reagents

Blood was used fresh or up to 2 weeks after collection from healthyvolunteers. Red blood cells (RBCs) and hemoglobin were prepared asdescribed previously (Huang et al., J Clin Invest 115(8):2099-2107,2005). All chemicals were purchased from Sigma (St. Louis, Mo.) unlessnoted otherwise. Visible absorbance spectra and kinetic data werecollected on Cary 50 and HP8453 UV-visible spectrophotometers (AgilentTechnologies, Palo Alto, Calif.) and with an SX20 Stopped-FlowSpectrometer (Applied Photophysics Limited, Leatherhead, UK). Allexperiments were performed in phosphate buffered saline (Sigma). Carbonmonoxide (CO)-saturated buffer was prepared by bubbling 20 mL of PBSwith CO gas for at least 15 minutes. Stock sodium dithionite solutionwas prepared by adding PBS degassed by Argon flow-through to a degassedvial of dry sodium dithionite.

Expression and Purification of Recombinant Neuroglobin

Recombinant neuroglobin (Ngb) H64Q protein was purified from E. colicultures from a modified method based on previous work (Tiso et al., JBiol Chem 286(20):18277-18289, 2011). SoluBL21 E. coli cells (Genlantis)containing the pET28-NgbH64Q plasmid were grown in TB broth supplementedwith 30 μg/ml Kanamycin. Expression was induced at OD_(600nm)=0.8 byadding 1 mM isopropyl-1-thio-β-D-galactopyranoside and carried out for24 hours at 37° C. 8-aminolevulinic acid (0.4 mM) was added at inductionto enhance the production of the heme cofactor. Cells were harvested andresuspended in lysis buffer (50 mM MOPS, pH 7.0, 1 mM EDTA, 1 mg/mllysozyme, 1 mM PMSF, 0.5 mM DTT) and lysed by sonication. Supernatantwas loaded into a DEAE-sepharose column equilibrated with buffer A (50mM MOPS pH 7.0, 10 mM NaCl). The samples were washed with 3 columnvolumes of buffer A and eluted by a linear gradient to 100% buffer B (50mM MOPS pH 7.0, 100 mM NaCl). Ngb fractions were pooled andconcentrated. For further purification, the concentrated samples wererun in a gel filtration column (Sephacryl S-200 HR, GE Healthcare).Purity was assessed by SDS-PAGE and UV-Vis spectroscopy.

Kinetics of Carboxylated Hb Mixed with Neuroglobin

Carboxylated Hb (HbCO) was prepared by adding an excess of sodiumdithionite to thawed Hb and mixing with CO-saturated buffer at a ratioof at least 4:1. Excess CO was removed by passing through a desaltingcolumn inside a glove box. For anaerobic experiments, an excess ofsodium dithionite was then added to the HbCO. Thawed Ngb-H64Q was mixedwith an excess of potassium ferricyanide and passed through a desaltingcolumn to obtain the oxidized form. In some instances, Ngb-H64Q wasalready stored in the oxidized form at −80° C. Deoxygenated Ngb-H64Q wasobtained by adding an excess of sodium dithionite to the oxidized form.For aerobic experiments, the oxygenated form was obtained by passing thedeoxygenated form through a desalting column under aerobic conditionsimmediately before mixing with HbCO.

For kinetics measured with the Cary 50 or the HP8453 spectrophotometer,HbCO inside a cuvette of 1 cm path length was placed in the cell holderand brought to either 25° C. or 37° C. The deoxygenated or oxygenatedNgb-H64Q was quickly equilibrated to the same temperature using a waterbath next to the spectrophotometer. Reaction was initiated by injectingNgb-H64Q into the HbCO solution for a final concentration of 40 μM ofboth proteins. Collection of absorbance was initiated with a delay of 1to 10 seconds and continued for up to 20 minutes as the mixture wascontinuously stirred. A final concentration of 1-5 mM of sodiumdithionite was present in anaerobic reactions. For kinetics measuredwith the SX20 Stopped-Flow Spectrometer, the sample lines of theinstrument were first washed with pure PBS for aerobic experiments andwith PBS containing 5 mM sodium dithionite for anaerobic experiments.The sample lines and syringes of the apparatus were equilibrated toeither 25° C. or 37° C. HbCO and Ngb-H6Q were then loaded into 2.5 mlsyringes of the apparatus that contained 5 mM sodium dithionite foranaerobic experiments. HbCO and Ngb-H64Q were mixed 1:1 with a dead timeof less than 2 msec for a final concentration of 25-30 μM of bothproteins. Absorbance of the reaction mixture was followed for no morethan 200 seconds.

Kinetics of Carboxylated RBCs Mixed with Neuroglobin

Red cells were obtained by washing 50-100 μL of blood with PBS 5 to 7times by centrifugation at 1000×g for 5-10 minutes. The washed red cellswere diluted in 1 to 2 ml of PBS and deoxygenated while on ice and slowstirring by a passing flow of argon gas for up to 1 hour. For anaerobicexperiments, argon was passed briefly and an excess of sodium dithioniteto Hb was added to the red cells. Carboxylated red cell-encapsulated Hbwas obtained by diluting the deoxygenated red cell solution with a ratioof at least 4:1. Excess CO was removed by washing the red cells 2 timeswith degassed PBS (containing 5-10 mM dithionite for anaerobicexperiments) by centrifugation for 5 minutes at 1000×g in degassed andseptum-capped 15 mL centrifuge tubes. After washing, the red cells wereresuspended to a final concentration of 100-200 μM, with an excess ofsodium dithionite for anaerobic experiments.

Oxygenated or deoxygenated Ngb-H64Q was prepared following the sameprocedure as that described for the experiments with pure Hb. In someexperiments, after initiating the reaction, red cells were separatedfrom Ngb-H64Q to measure absorbance spectra. In this case, the reactiontemperature was regulated with an ISOTEMP™ stirring hotplate and waterbath combination (Fisher Scientific). Red cell-encapsulated HbCO andoxygenated or deoxygenated Ngb-H64Q were equilibrated to 25° C. or 37°C. in separate glass vials. Reaction was initiated by injecting Ngb-H64Qinto the red cell solution for a final concentration of 40 μM of bothproteins. An equivalent volume of PBS (with or without dithionite) wasinjected into a control sample of carboxylated red cells. Periodically,0.5 ml of the reaction and the control sample were taken and centrifugedfor 30-60 seconds at 5000×g in 1.5 mL microcentrifuge tubes. Thesupernatant containing Ngb-H64Q was removed (5 mM sodium dithionite wasadded in aerobic experiments to prevent auto-oxidation of the protein)and stored on ice.

A solution of 0.5% NP40 in PBS (always containing 5 mM sodium dithionitefor anaerobic experiments and sometimes for aerobic) was added to thered cell pellet to lyse the cells. Hb absorbance in the lysed red cellsolution was measured with the Cary 50 spectrophotometer in a 1 cm pathlength cuvette. This cycle was repeated every 1.5-5 minutes for a totalof six times, giving six absorbance measurements of the Hb. The controland reaction samples were continuously stirred. The time when absorbanceof hemoglobin was measured in the reaction was assumed to be the timeelapsed after injection of Ngb-H64Q to 15 or 30 seconds after the startof centrifugation (for 30 or 60 second centrifugation durations,respectively). After the last (6^(th)) time point was measured,absorbance of the stored supernatant samples of the reaction and controlmixtures was recorded as well. In some experiments, the red cells werenot separated from Ngb-H64Q; instead, absorbance of the whole mixturewas recorded with the Integrating Sphere attachment of a Cary 100spectrophotometer. This setup collects light scattered by the red cells,thereby providing absorbance spectra sufficiently accurate for spectraldeconvolution. The procedure for these experiments was the same as thatfor mixing Ngb-H64Q with pure HbCO in the Cary 50, after preparation ofcarboxylated red cells.

Least Squares Deconvolution

Standard reference spectra of the oxidized (met), deoxygenated (deoxy),oxygenated (02) and carboxylated (CO) forms of hemoglobin (Hb) andneuroglobin H64Q (Ngb-H64Q) were obtained. After thawing protein on ice,spectra of the oxidized form were obtained by mixing with an excess ofpotassium ferricyanide and passing through an ECONO-PAC™ 10 DG DesaltingColumn (Bio-Rad Laboratories, Hercules, Calif.). Spectra of deoxygenatedspecies were recorded after adding an excess of sodium dithionite to theoxidized form. Spectra of the oxygenated form were recorded immediatelyafter passing deoxygenated species through the desalting column underaerobic conditions. Spectra of the carboxylated form were measured aftermixing the deoxygenated species with CO-saturated buffer in a ratio of1:4. All standard spectra were collected at 20° C., 25° C., and 37° C.on the Cary 50 spectrophotometer.

Deconvolution of experimental spectra was performed with a least-squaresfitting routine in Microsoft Excel. Because the change in absorbance ofthe kinetic experiments is not great, all spectra composed of both Hband Ngb-H64Q were always fit between 450 and 700 nm, 490 and 650 nm, and510 and 600 nm, with and without constraining the Hb and Ngb-H64Qconcentrations to be equal to each other, in order to confirm theaccuracy of the deconvolution. Absorbance spectra from anaerobicexperiments were deconvoluted using carboxylated and deoxygenatedstandards of Hb and Ngb-H64Q. Absorbance spectra from aerobicexperiments were deconvoluted using the standards of the oxidized,carboxylated and oxygenated forms of Hb and Ngb-H64Q. For the red cellexperiments where Hb was separated from Ngb-H64Q and dithionite wasafterwards added to either red cells in aerobic experiments or to thesupernatant in anaerobic experiments, deoxygenated standards were usedin deconvolution instead of the oxygenated and oxidized forms. Beforedeconvoluting spectra collected with the Stopped-Flow spectrometer, andsometimes those with the HP8453, absorbance values were remapped to thesame wavelengths as those used by the Cary 50 spectrophotometer usingthe interp1 function of MATLAB™, employing piecewise cubic hermiteinterpolation.

Results

The reaction of carboxylated red blood cells (RBCs) with either bufferor a solution of deoxy H64Q neuroglobin was studied (FIG. 1). When theRBCs were mixed with buffer, no appreciable change in the ratio of HbCOoccurred (FIG. 1, left panel). When the cells were mixed with thedeoxyNgb mutant, all CO was removed from the RBCs within about 15minutes. The changes in the neuroglobin fraction are consistent with atransfer of CO from Hb to Ngb.

Another experiment with similar conditions is shown in FIG. 2. Thereaction was completed in just over 10 minutes, consistent with theprevious results shown in FIG. 1.

In addition, the reaction of carboxylated red blood cells with oxy H64Qneuroglobin was evaluated (FIG. 3). As observed for the deoxyNgb, theoxygenated form is also able to scavenge CO from the red blood cells atan even faster rate than that observed in the previous experiment. Theprocess is completed in less than 5 minutes. The changes in theneuroglobin fraction are consistent with a transfer of CO from Hb toNgb.

Altogether, these results indicate that neuroglobin is able to remove COfrom carboxylated hemoglobin that is located inside red blood cells(across compartment) within 5-10 minutes, a time scale suitable forclinical treatment.

Example 2 Auto-Oxidation Rates for WT and Mutant Neuroglobins

Ferrous-dioxygen complexes in heme proteins have intrinsic rates ofauto-oxidation, where the oxygen molecule can take an electron from theheme iron to form oxidized heme and superoxide radical:

Auto-oxidation is a side reaction that can be detrimental for theintended neuroglobin applications (e.g., binding and removing CO fromhemoglobin). For the purpose of treating carboxyhemoglobinemia,neuroglobin is infused as a ferrous dioxygen complex (oxy form, Fe²⁺—O₂)that is able to: i) bind CO and ii) deliver oxygen to the tissues.Oxidation of the oxyneuroglobin leads to formation of the ferric form(met form, Fe³⁺) that cannot accomplish either of the two functionsmentioned above. The reaction also forms the radical superoxide species(O₂ ⁻.) that would cause increased oxidative stress.

The auto-oxidation rates for wild-type and several neuroglobin His64mutants were determined and are summarized in Table 2.

TABLE 2 Auto-oxidation rates for wild-type neuroglobin and mutantsK_(ox) t_(1/2) (min⁻¹) (min) Human Neuroglobin, wild-type 0.230 ± 0.0303.0 Human Neuroglobin, H64W 0.076 ± 0.006 9.1 Human Neuroglobin, H64A0.066 ± 0.005 10.5 Human Neuroglobin, H64L 0.010 ± 0.004 69.3 HumanNeuroglobin, H64Q 0.010 ± 0.002 69.3 Values determined at 37° C. insodium phosphate 100 mM, pH 7.4. t_(1/2); calculated half-life of theferrous dioxygen complex.

Wild-type neuroglobin has a fast auto-oxidation rate; half of anyexisting ferrous-dioxygen complex is oxidized every 3 minutes. However,the neuroglobin mutants H64L and H64Q have auto-oxidation rates 23-foldslower than the wild type protein. Based on these findings, the H64Qmutant was selected for the in vivo studies described in Example 3.

Example 3 Recombinant H64Q Neuroglobin is an Antidote forCarboxyhemoglobinemia in Vivo

This example demonstrates that administration of a recombinant globinmolecule that binds carbon monoxide with high affinity efficientlyclears HbCO from the blood of CO-exposed mice and thus represents anantidote for carboxyhemoglobinemia and/or carbon monoxide poisoning. Inthe experiments described below, the CO is removed from red blood cellsafter a five minute infusion of recombinant mutant neuroglobin H64Q. TheH64Q mutant neuroglobin used in the studies below also containsmutations at three cysteine residues (C46G/C55S/C120S).

In the following experiments, H64Q neuroglobin was administered at adose ranging between 13.6 and 27.2 mg (4-8 mM). Blood volume in humansis approximately 5000× greater, therefore this dose range is equivalentto a human dose range of 68-136 grams.

The capacity of recombinant H64Q neuroglobin to remove HbCO from bloodin vivo was evaluated in CO-exposed mice. Mice were exposed to 1500 ppmCO for 60-70 minutes, CO was then stopped and either PBS (200 μl) orconcentrated H64Q neuroglobin (200 μl) was infused for 5 minutes(PBS—FIG. 4; H64Q neuroglobin—FIG. 6) or 10 minutes (H64Qneuroglobin—FIG. 5). Blood samples (approximately 10 μl) were drawnevery 5-10 minutes, the red cells were washed, lysed and chemicallyreduced with dithionite and monitored for HbCO and NgbCO. This assayshows the amount of CO that is in the red blood cell before and afterthe H64Q NgB infusion in vivo. As shown in FIGS. 5 and 6, infusion ofH64Q neuroglobin rapidly removes HbCO from the blood within the 5 or 10minute infusion period.

The decay in concentration of HbCO in the blood of CO-exposed mice aftera five-minute infusion of PBS or H64Q neuroglobin is shown in FIGS. 7Aand 7B. FIG. 7A shows absolute HbCO levels over time and FIG. 7B showsthe relative change in HbCO level over time. The results shown in thesefigures clearly demonstrate that infusion of H64Q neuroglobin leads to amore rapid clearance of HbCO from the blood, and a greater relativechange in HbCO over time, than infusion with PBS. Similarly, FIG. 8shows that infusion of H64Q for either 5 or 10 minutes leads to agreater percent HbCO decay compared with infusion of PBS for the sameperiod of time.

To confirm that neuroglobin is cleared as NgbCO in the urine of infusedmice, absorbance of urine from bladders of mice sacrificed approximately75 minutes after the end of CO exposure was determined. The results areshown in FIG. 9A. The top three traces show the absorbance due toneuroglobin, which include 82 to 91% NgbCO. The bottom traces indicatethe absorbance that is not attributable to Ngb. In addition, thepresence of NgbCO in the urine of CO-exposed mice was confirmed by thepresence of red-colored urine in these mice (see syringe in FIG. 9B).

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1. A method of treating carboxyhemoglobinemia in a subject, comprising: selecting a subject with carboxyhemoglobinemia; and administering to the subject a therapeutically effective amount of a recombinant globin molecule that binds carbon monoxide with high affinity, wherein the recombinant globin molecule comprises: a human recombinant neuroglobin with a H64Q, H64L, H64A or H64W mutation; or a human recombinant cytoglobin with a H81Q, H81A, H81L or H81W mutation, thereby treating carboxyhemoglobinemia in the subject.
 2. The method of claim 1, wherein the human recombinant neuroglobin further comprises a C46G mutation, a C55S mutation, a C120S mutation, or any combination thereof.
 3. The method of claim 2, wherein the human recombinant neuroglobin comprises the amino acid sequence of SEQ ID NO:
 3. 4. The method of claim 1, wherein the human recombinant cytoglobin further comprises a C38S mutation, or a C83S mutation, or both a C38S and a C83S mutation.
 5. The method of claim 4, wherein the human recombinant cytoglobin comprises the amino acid sequence of SEQ ID NO:
 6. 6. The method of claim 1, wherein the subject has at least 3% carboxyhemoglobin in their blood.
 7. The method of claim 1, wherein the recombinant globin molecule is administered by intravenous infusion.
 8. (canceled)
 9. A method of removing carbon monoxide from hemoglobin in blood or tissue, comprising contacting the blood or tissue with a recombinant globin molecule that binds carbon monoxide with high affinity, wherein the recombinant globin molecule comprises: a human recombinant neuroglobin with an H64Q, H64L, H64A or H64W mutation; or a human recombinant cytoglobin with an H81Q, H81A, H81L or H81W mutation, thereby removing carbon monoxide from hemoglobin in the blood or tissue.
 10. The method of claim 9, wherein the human recombinant neuroglobin further comprises a C46G mutation, a C55S mutation, a C120S mutation, or any combination thereof.
 11. The method of claim 10, wherein the human recombinant neuroglobin comprises the amino acid sequence of SEQ ID NO:
 3. 12. The method of claim 9, wherein the human recombinant cytoglobin further comprises a C38S mutation, or a C83S mutation, or both a C38S and a C83S mutation.
 13. The method of claim 12, wherein the human recombinant cytoglobin comprises the amino acid sequence of SEQ ID NO:
 6. 14. The method of claim 9, which is an in vitro method.
 15. The method of claim 9, which is an in vivo method, wherein contacting the blood or tissue with a recombinant globin molecule comprises administering the recombinant globin molecule to a subject.
 16. The method of claim 15, wherein the subject has at least 5% carboxyhemoglobin in their blood.
 17. The method of claim 15, wherein the recombinant globin molecule is administered by intravenous infusion.
 18. (canceled)
 19. A human recombinant globin molecule comprising: a human recombinant neuroglobin comprising a mutation at residue 64, and further comprising a C46G mutation, a C55S mutation and a C120S mutation; or a human recombinant cytoglobin comprising a mutation at residue 81, and further comprising a C38S mutation and a C83S mutation.
 20. The human recombinant globin molecule of claim 19, wherein the mutation at residue 64 of the human recombinant neuroglobin is a H64Q, H64L, H64A or H64W mutation; or wherein the mutation at residue 81 of the human recombinant cytoglobin is a H81Q, H81A, H81L or H81W mutation.
 21. The human recombinant globin molecule of claim 20, comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:
 6. 22-24. (canceled)
 25. A composition comprising the human recombinant globin molecule of claim 19 and a pharmaceutically acceptable carrier. 